Phosphorylation of proteins on serine, threonine, and tyrosine residues is the most common form of reversible protein modification in eukaryotic cells. Protein phosphorylation is known to be an important means of regulating a wide variety of cellular processes, including cell division and differentiation, cell motility, muscle contraction, protein synthesis, and cellular metabolism. Many disease processes are now known to be the result of dysregulated protein phosphorylation including many forms of cancer and heart disease, diabetes and other metabolic diseases, and diseases of muscle such as muscular dystrophy. Thus, a detailed understanding of the biochemical properties of the enzymes that catalyze the phosphorylation of proteins, the protein kinases, is critical for understanding many important disease processes and will be critical to developing therapeutic strategies that target specific protein kinases. A key to understanding how protein kinases function in living cells will be to determine their biochemical properties in situ. For most protein kinases, however, it is currently impossible or quite difficult to measure their activity in living cells. The overall goal of the proposed research is to apply recent experimental findings to the development of a new technique for measuring protein kinase activities in living cells using fluorescent protein kinase substrates. To achieve this goal, we have defined the following specific aims: Specific Aim 1: Develop fluorescent phosphate-acceptor substrates for three protein kinases, protein kinase C (PKC), the cAMP-dependent protein kinase (PKA), and the mitogen-activated protein (MAP) kinase known as ERK. These three protein kinases are key enzymes involved in regulating cell growth and differentiation. Specific Aim 2: Using single cell microinjection techniques, incorporate these fluorescent kinase substrates into cultured cells and follow in situ activation of the protein kinases in response to appropriate cell stimuli. There are many applications of this technology including determining whether a specific protein kinase is involved in regulating a particular cellular functions, testing potential therapeutic agents for their action on specific protein kinases, and determining the possible roles of specific protein kinases in various disease processes. Thus, a general method for assaying protein kinase activities in cultured cells could be applied to a variety of experimental problems directed towards understanding the role of specific protein kinases in physiological and pathophysiological processes.